The Complex Airway Epithelial Apparatus and its Role in Health and Disease
The human lung is suited for providing gas exchange from the atmosphere to the body: with every breath, oxygen enters the bloodstream, and carbon dioxide is removed. This constant environmental exposure makes the defense systems of the lung extremely important in maintaining health and preventing disease. Specifically, the surface epithelial cells which line the lung are protected by a tightly regulated layer of mucus which functions to entrap pathogens and inhaled particulates. These cells also contain tiny hair-like projections called cilia which propel the semi-liquid mucus gel layer out of the lung. This system, commonly called the mucociliary clearance (MCC) apparatus, facilitates the lung to entrap and clear particles and pathogens which enter the lung from the environment. Cilia are complex in structure, with outer and inner rings of microtubules which propel the cilia in specific beat patterns that are also coordinated with one another. Other parts of the apparatus are similarly complex, including the content and molecular makeup and the electrolyte and water content of the mucus gel layer, which determine its physical characteristics and transportability. When the mucociliary clearance apparatus is impaired, whether due to malformation or dysfunction of cilia, dysregulation of the ion and water transport, abnormalities of the mucus itself, or other insults, lung disease can result.
Many Diseases Linked to Ciliary Dysfunction \
Many diseases are affected by dysfunction of the functional microanatomy of the airway and consequently the mucociliary clearance apparatus. For example, cystic fibrosis (CF) is the most common lethal genetic disease in the Caucasian population, and is a significant cause of morbidity and early mortality from progressive lung disease. (See Rowe S M, et al., Cystic fibrosis, N Engl J Med 2005; 352:1992-2001.) About 30,000 children and adults in the United States are affected by CF and the prevalence is estimated at 70,000 worldwide. Further, mild diseases due to partial abnormalities in the causative CF protein, termed cystic fibrosis transmembrane regulator (CFTR), are about 10-fold more common than typical forms of the disease. It is well established that the primary defect in CF, dysfunction of the CFTR protein, results in abnormal mucociliary clearance (MCC) due to the absence of chloride and bicarbonate transport, and is associated with dysregulation of the airway surface liquid (ASL) and periciliary liquid layer (PCL) depths. As another example, primary ciliary dyskinesia (PCD) is a disorder in which structural ciliary defects result in abnormal ciliary motion, which in turn leads to impaired mucociliary clearance and susceptibility to recurrent sinopulmonary infections. (See Bush A et al. “Primary ciliary dyskinesia: current state of the art. Archives of disease in childhood”, 2007; 92:1136-40). Chronic obstructive pulmonary disease (COPD), recently the third leading cause of death in the U.S., is also characterized by mucus stasis and impaired mucociliary clearance. Other common lung diseases are also affected by dysfunction of the epithelial surface, including, but not limited to, types of interstitial lung disease such as its most common form idiopathic pulmonary fibrosis which are characterized by abnormal function of the surface mucins, the proteins that form the mucus gel.
Even people with normal epithelial function and a normally functioning cellular mucociliary clearance apparatus during health can also be impacted by difficulty with impaired mucus clearance and increased mucus production. For example, individuals with neuromuscular weakness caused by congenital or genetic conditions, such as, but not limited to, muscular dystrophy, spinal muscular atrophy, and amyotrophic lateral sclerosis, suffer with recurrent pneumonia due to poor cough clearance which leads to mucous stasis. In addition, individuals with acquired anatomic problems resulting in muscular weakness, such as but not limited to, paraplegia, quadriplegia, diaphragmatic paralysis and the like, suffer the same fate. Other subjects, such as those suffering from excess mucus production due to conditions such as, but not limited to, asthma and status asthmaticus, those suffering from impaired immunity due to conditions such as, but not limited to, immunoglobulin deficiency, SCID, hyper-IgE syndrome, and similar conditions, those suffering from anatomic respiratory abnormalities impairing mucus clearance, those suffering from recurrent pneumonia for unclear causes and those suffering from oropharyngeal abnormalities, suffer from atelectasis and/or pneumonia due to excess mucus production that overwhelms the capacity of the mucociliary clearance apparatus to transport it effectively. These disorders due to impaired mucous clearance and/or excess mucous production has been a serious recurrent problem causing considerable morbidity and are also a contributing cause to mortality.
The Role of Rheology in the Study of Disease
Mucus itself can be characterized in part by its viscosity, or its resistance to physical flow. Thicker, more viscous mucus is more difficult for the mucociliary apparatus to clear, contributing to disease. The study of viscosity by rheology measurements allows for characterizing mucus physical properties, understanding mechanisms of human disease, and evaluating the effect of therapeutics to address abnormal mucus. May studies have shown that expectorated sputa from CF patients are abnormal, demonstrating a highly viscous nature and increased percentage of solid content. (See Serisier D J et al., “Macrorheology of cystic fibrosis, chronic obstructive pulmonary disease & normal sputum”, Respiratory research 2009; 10:63; Chernick W S and Barbero G J, “Composition of tracheobronchial secretions in cystic fibrosis of the pancreas and bronchiectasis”, Pediatrics 1959; 24:739-45; Matsui H et al., “Reduced three-dimensional motility in dehydrated airway mucus prevents neutrophil capture and killing bacteria on airway epithelial surfaces”, J Immunol 2005; 175:1090-9; Dawson M at al., “Enhanced viscoelasticity of human cystic fibrotic sputum correlates with increasing microheterogeneity in particle transport”, J Biol Chem 2003; 278:50393-401; and Martens C J et al., “Mucous Solids and Liquid Secretion by Airways: Studies with Normal Pig, Cystic Fibrosis Human, and Non-Cystic Fibrosis Human Bronchi”, American journal of physiology Lung cellular and molecular physiology 2011) Prior studies have also suggested that COPD sputum has increased viscosity. (See Redding G J et al. “Physical and transport properties of sputum from children with idiopathic bronchiectasis”, Chest 2008; 134:1129-34). Mucus is also characterized by its adherence. Abnormal adherence to the surface structures of the airway are thought to substantially contribute to clinical disease.
Limitations of Current Methods
Certain methods for investigating the functional microanatomy of the airway surface in natural, untreated airway epithelia, including cell and tissue culture systems and in vivo methods, are limited. Current knowledge of respiratory cilia and PCL morphology is based on electron microscopy. (Matsui H. et al., “Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease”, Cell 1998; 95:1005-15; and Sanderson M J and Sleigh M A, “Ciliary activity of cultured rabbit tracheal epithelium—beat pattern and metachrony”, Journal of Cell Science 1981; 47:331-47.) These methods only provide static images of epithelia when they are not functioning; fixation and harsh processing are likely to alter cilia and PCL morphology, and could account for disparities in the conclusions on the role of ASL/PCL in CF and other lung diseases. ASL height can be measured in vitro using confocal microscopy by aid of fluorescent staining, but is technically challenging, difficult to achieve the high axial resolution required to accurately assess ASL/PCL, and prone to artifacts caused by interference or removal of the native fluid and flow by the exogenous contrast agents. (See also Randell S H, and Boucher R C, “Univ NCVLG. Effective mucus clearance is essential for respiratory health”, American Journal of Respiratory Cell and Molecular Biology 2006; 35:20-8). Measuring mucociliary transport (MCT) by tracking fluorescent beads is problematic because the beads are known to cause the mucus to agglomerate and significantly slow transport rates. In addition to these limitations, spatial and temporal correlation are very difficult with these assays, as measurement of ciliary beat frequency (CBF), ASL, PCL and MCT are generally done at different time points with different imaging modalities, processing methods, and cells. Likely, none of these methods are suitable for use in vivo, including both human testing and experimental animals to characterize the effect of drugs. Since these parameters are closely interrelated and can influence each other, it is essential to monitor these parameters simultaneously to gain a full understanding of the functional airway.
Accordingly, it may be beneficial to address at least some of the above-described deficiencies.
Unique Advantages of μOCT
Techniques for reflectance microscopy in vivo have recently been introduced for the visualization of tissue microstructure at architectural and cellular levels. These include optical coherence tomography (OCT) which has been developed to provide unprecedented cellular detail and live motion capture. (See Tearney G J et al., “In vivo endoscopic optical biopsy with optical coherence tomography”, Science 1997; 276:2037-9; Fujimoto J G et al., “Optical coherence tomography: An emerging technology for biomedical imaging and optical biopsy”, Neoplasia 2000; 2:9-25; Drexler W et al., “In vivo ultrahigh-resolution optical coherence tomography”, Optics Letters 1999; 24:1221-3; Gabriele M L et al., “Peripapillary nerve fiber layer thickness profile determined with high speed, ultrahigh resolution optical coherence tomography high-density scanning”, Investigative Ophthalmology & Visual Science 2007; 48:3154-60; Srinivasan V J et al., “Noninvasive volumetric Imaging and morphometry of the rodent retina with high-speed, ultrahigh-resolution optical coherence tomography”, Investigative Ophthalmology & Visual Science 2006; 47:5522-8; Wojtkowski M. et al., “Three-dimensional retinal imaging with high-speed ultrahigh-resolution optical coherence tomography”, Ophthalmology 2005; 112:1734-46; Tearney G J et al., “In vivo endoscopic optical biopsy with optical coherence tomography”, Science 1997; 276:2037-9; and Vakoc B J et al. “Three-dimensional microscopy of the tumor microenvironment in vivo using optical frequency domain imaging”, Nat Med 2009; 15:1219-23). The technology uses the reflectance signature of near-infrared light to permit real-time imaging with cellular level detail, and has been employed successfully for microscopic analysis of coronary artery and esophageal mucosa by the endoscopic approach in living human subjects. OCT uses coherence gating for optical sectioning to attain an axial resolution or section thickness ranging from 1-10 μm. (See Yun S H et al., “Comprehensive volumetric optical microscopy in vivo”, Nat Med 2006; 12:1429-33; Jang I K et al., “Visualization of coronary atherosclerotic plaques in patients using optical coherence tomography: Comparison with intravascular ultrasound”, Journal of the American College of Cardiology 2002; 39:604-9; Yabushita H. et al., “Characterization of human atherosclerosis by optical coherence tomography”, Circulation 2002; 106:1640-5; Tearney G J et al., “Quantification of macrophage content in atherosclerotic plaques by optical coherence tomography”, Circulation 2003; 107:113-9; MacNeill B D et al., “Focal and multi-focal plaque distributions in patients with macrophage acute and stable presentations of coronary artery disease”, Journal of the American College of Cardiology 2004; 44:972-9; Jang I K et al., “In vivo characterization of coronary atherosclerotic plaque by use of optical coherence tomography”, Circulation 2005; 111:1551-5; Vakoc B J et al., “Comprehensive esophageal microscopy by using optical frequency-domain imaging (with video)”, Gastrointestinal Endoscopy 2007; 65:898-905; Yun S H et al., “Comprehensive volumetric optical microscopy in vivo”, Nature Medicine 2006; 12:1429-33; Poneros J M et al., “Diagnosis of specialized intestinal metaplasia by optical coherence tomography”, Gastroenterology 2001; 120:7-12; and Evans J A et al., “Mino-Kenudson M, Nishioka N S, Tearney G J. Optical coherence tomography to identify intramucosal carcinoma and high-grade dysplasia in Barrett's esophagus”, Clinical Gastroenterology and Hepatology 2006; 4:38-43).
Since OCT is not reliant on a high numerical aperture lens, it can employ an imaging lens with a relatively large confocal parameter, facilitating a greater penetration depth (about 1 mm) and a cross-sectional display format. OCT is particularly well suited for non-invasive microscopy in cells and tissues since it can be implemented via small, flexible probes, does not require contact with the cell surface or use of contrast medium, and acquires high resolution images with very rapid acquisition times and flexible focal range.
An acquisition of the OCT signal in the wavelength domain as opposed to the time domain can provide orders of magnitude improvement in imaging speed while maintaining excellent image quality. One such second-generation imaging technology is termed micro-OCT (μOCT). (See de Boer J F et al., “Improved signal-to-noise ratio in spectral-domain compared with time-domain optical coherence tomography”, Optics Letters 2003; 28:2067-9; Choma M A et al., “Sensitivity advantage of swept source and Fourier domain optical coherence tomography”, Optics Express 2003; 11:2183-9; Nassif N. et al., “In vivo human retinal imaging by ultrahigh-speed spectral domain optical coherence tomography” Optics Letters 2004; 29:480-2; and Yun S H et al., “High-speed wavelength-swept semiconductor laser with a polygon-scanner-based wavelength filter”, Optics Letters 2003; 28:1981-3).
With μOCT, high-resolution ranging is conducted in tissue by detecting spectrally resolved interference between the tissue sample and a reference. (See also Wojtkowski M. et al., “In vivo human retinal imaging by Fourier domain optical coherence tomography”, J Biomed Opt 2002; 7:457-63). Since μOCT can utilize a high-speed linear camera, it is capable of capturing images at more than 50 million pixels per second, which is approximately two orders of magnitude faster than conventional time-domain OCT systems. (See also Wojtkowski M. et al., “Three-dimensional Retinal Imaging with High-Speed Ultrahigh-Resolution Optical Coherence Tomography”, Ophthalmology 2005). By using light sources with large spectral bandwidths (˜150 nm), recent studies have shown that μOCT images can be obtained in vivo with an axial resolution of approximately 2 μm, which is adequate to visualize the PCL, beating cilia, and mucosal glands. (See Gabriele M L et al., “Peripapillary nerve fiber layer thickness profile determined with high speed, ultrahigh resolution optical coherence tomography high-density scanning”, Invest Ophthalmol Vis Sci 2007; 48:3154-60; Srinivasan V J et al., “Noninvasive volumetric imaging and morphometry of the rodent retina with high-speed, ultrahigh-resolution optical coherence tomography”, Invest Ophthalmol Vis Sci 2006; 47:5522-8; and Wojtkowski M et al., “Three-dimensional retinal imaging with high-speed ultrahigh-resolution optical coherence tomography”, Ophthalmology 2005; 112:1734-46). With this acquisition rate and resolution, μOCT is a very powerful tool for investigating the respiratory mucosa.
Status of Screening and Secondary Characterization
High throughput screening (HTS) for exemplary modulators of epithelial function has been successful as a drug discovery modality, identifying certain small molecules, biologics, and pathways relevant to human airway disease. While this is been particularly successful in CF to identify modulators of CFTR, the assay systems typically used are limited in scope, and cannot directly interrogate epithelial function relevant to mucociliary transport in humans. Rather, these approaches are reductionist towards specific pathways that may or may not be directly relevant to a broad array of human diseases. For example, almost all HTS technologies for CF attempt to identify alterations in chloride, halide, or sodium transport, and can only probe one of these pathways depending on the specific probe. This reductionist approach makes the assay limited in scope, and is relevant only to diseases where that ion transport pathway is relevant.
Accordingly, there is a need to address and/or at least some of the deficiencies described herein above.